Field of the Invention
The present invention relates to culture methods for the proliferation of pluripotent stem cells while maintaining an undifferentiated state, and particularly relates to a culture method for stable proliferation of human pluripotent stem cells under serum-free, feeder-free conditions and under single seeding conditions while maintaining an undifferentiated state and the like.
Discussion of the Background
Pluripotent stem cells such as ES (Embryonic stem) cells, iPS (induced pluripotent stem) cells and the like are expected to be usable for regenerative medicine and the like in view of the superior proliferativity and pluripotency. In particular, iPS cells are considered a highly superior material for regenerative medicine, since production and obtainment are relatively easy, there are few ethical restrictions for production, and further, from the aspect of rejection in transplantation.
These pluripotent stem cells have conventionally been cultured by co-culture with a carrying cell (hereinafter a feeder cell) such as a fibroblast and the like in a medium containing serum. For example, in Cell, 2006, 126, 663-76, which is incorporated herein by reference in its entirety and which is the world-first report by Yamanaka et al. on the production of iPS cells, iPS cells were established and maintained and proliferated under conditions using a feeder cell and serum. These pluripotent stem cells proliferate while forming a colony where individual cells are clustered. When a colony is dissociated into single cells and used for seeding (hereinafter single cell-seeding), the cells become unstable. It is therefore a general practice to seed a colony maintained to have a certain size (hereinafter colony seeding). For example, Nature Communications, 2012, 3:1236, which is incorporated herein by reference in its entirety, discloses an example showing that the proliferation conditions of cells are prone to influence from culture environment in the case of single cell-seeding compared to colony seeding. In other words, single cell-seeding poses higher culture difficulty than colony seeding.
To perform feeder-free culture, it is necessary to coat the bottom of a culture vessel with a substrate or scaffolding material replacing a feeder cell. As the substrate, an extracellular matrix component is often used. JP-A-2011-78370, which is incorporated herein by reference in its entirety, discloses that use of an active fragment of laminin 511 as a substrate is preferable for the proliferation of human ES/iPS cell, and single cell-seeding is also possible.
WO 2012/019122 and Nature Methods, 2011, 8, 424-429, both of which are incorporated herein by reference in their entireties, disclose the composition of a serum-free medium for human pluripotent stem cells. This composition called E8 contains DMEM/F12 as a basal medium, and further contains some factors such as bFGF, insulin and the like. At present, it is considered the minimum composition for cultivating human pluripotent stem cells.
Ethanolamine is known to contribute to the promoted proliferation of mesenchymal stem cells when used as an additive in a medium. For example, patent document 3 patent document 3: JP-A-2006-325445, which is incorporated herein by reference in its entirety, discloses an example suggesting that ethanolamine promotes proliferation of mesenchymal stem cells.
In addition, JP-A-2009-542247, which is incorporated herein by reference in its entirety, describes a method of maintaining primate embryonic stem cells in a medium containing ethanolamine, 2-mercaptoethanol, a complex of oleic acid with fatty acid-free bovine albumin, heparin and the like, and the like, and WO 2005/063968, which is incorporated herein by reference in its entirety, describes a medium for culturing ES cells, which contains 2-mercaptoethanol, 2-ethanolamine, a complex of oleic acid with fatty acid-free bovine serum albumin and the like. U.S. Pat. No. 8,569,061, which is incorporated herein by reference in its entirety, discloses a medium for ES cells, which contains human albumin, ethanolamine, β-mercaptoethanol and the like.
The above-mentioned JP-A-2009-542247, WO 2005/063968, and U.S. Pat. No. 8,569,061 describe given amounts (10 μM, 10 μM, 100 μM, respectively) of 2-mercaptoethanol (β-mercaptoethanol) as an essential component of medium. In JP-A-2009-542247, oleic acid is added to bovine albumin after removal of fatty acid, such that the bovine albumin carries oleic acid, and 9.4 mg/g of oleic acid is added to albumin.
In the meantime, it has been reported heretofore that sulfated polysaccharides have an effect to protect growth factors from degradation, denaturation, inactivation and the like. For example, WO 92/13526, which is incorporated herein by reference in its entirety, discloses that carrageenan stabilizes bFGF, and describes in the Examples that a protecting agent containing sulfated polysaccharides such as heparin, dextran sulfate, carrageenan and the like protects bFGF from hydrolysis and heat denaturation. However, an effect provided by a combination of ethanolamine and sulfated polysaccharides is not disclosed. Moreover, the above-mentioned JP-A-2009-542247 discloses a medium integrally containing ethanolamine and heparin; however, a detailed effect of each of them has not been known to date.